The present invention relates to methods for UV illumination of blood or a blood-derived product for the purposes of reduction of pathogens therein. More specifically, the present invention describes methods of reducing damage to blood components caused by oxygenation and generation of reactive oxygen species (ROS) during UV illumination.
A major concern in the transfusion of donated, stored whole human blood or the various blood-derived products such as blood cells or protein fractions isolated from whole blood is the presence of pathogens including viral or bacterial contamination as well as a variety of other harmful microorganisms such as parasites, molds, and yeasts. Of particular concern are the blood-borne viruses that cause hepatitis (especially hepatitis A, hepatitis B, and hepatitis C) and acquired immune deficiency syndrome (AIDS). While any number of cell washing protocols may reduce the viral contamination load for samples of blood cells by physical elution of the much smaller virus particles, such washing alone is insufficient to reduce viral contamination to safe levels. In fact, some viruses are believed to be cell-associated, and unlikely to be removed by extensive washing and centrifugal pelleting of the cells. Current theory suggests that safe levels will ultimately require at least a 5 log (5 orders of magnitude) demonstrated reduction in infectious viral titer for cellular blood components. This 5 log threshold may be even greater for plasma, protein components, especially the clotting factors (Factor VIII, Factor IX) that are administered throughout the life of some hemophilia patients.
All blood collected in the United States is now screened for a number of infectious agents including HIV-1, HIV-2, HTLV-1, hepatitis B virus, hepatitis C virus and syphilis. Additionally, donors are screened for risk factors, and potential donors are eliminated that are considered at risk for the HIV virus. This makes blood transfusion a very safe procedure. Despite these practices, there is still a risk of becoming infected by a potentially deadly virus or bacteria via the transfusion of blood or blood products. Screens for contaminants are by nature not foolproof. There is also the quite likely occurrence of new infectious agents that enter the blood supply before the significance of the event is even known.
The use of pathogen reduction technologies has the potential of eliminating the remaining risks of transmission of infectious disease as a result of blood transfusion. Various approaches have been used to sterilize blood components with the most promising being the photochemical ones, two of which were approved by regulatory agencies for pathogen reduction in platelet concentrates. The Intercept method employs a psoralen and UVA light illumination while the Mirasol method uses riboflavin and UVA+UVB light illumination.
Short wavelengths ultraviolet light (UVC, 180-290 nm) is a known sterilizing agent that targets the nucleic acids of microorganisms. It has been used for pathogen reduction in optically-transparent biological fluids such as plasma and platelet concentrates. However, in opaque biological fluids such as red cell concentrates as well as in whole blood, UVC penetration is very limited due to absorption of UVC illumination by the red cells. As a result, all attempts to use UVC illumination for sterilizing whole blood or red cells have been unsuccessful so far.
Exposure of a complex biological system such as blood to UVC results in a number of photochemical reactions that lead to degradation of blood proteins. These reactions can be divided into direct and indirect effects. Direct effects of UVC on proteins result from absorption of photons by amino acids with a relatively high absorption coefficients in the UVC range of the spectrum. These include tryptophan, phenylalanine, proline, methionine and cystein. Photon absorption results in electronic excitation of the molecule. The excited molecule can either undergo a chemical change or relax back to its ground state.
Indirect effects involve transfer of the excitation energy from a molecule to a nearby oxygen molecule resulting in formation of reactive oxygen species (ROS). ROS are strongly oxidizing agents that can react with proteins and degrade their function.
The need therefore exists for methods of donor blood preservation and pathogen inactivation in blood or blood-derived products which minimize oxidative damage to blood components caused by formation of ROS due to illuminating with UVC light.